The Puregene Tissue Kit is a liquid-based kit for isolation of genomic DNA from a variety of samples (blood, plant, yeast, bacteria, cells, animal tissue and even bones). In this method, only non-toxic detergents and salts are used to isolate DNA by salt precipitation (as published in Sambrock, Fritsch, Maniatis - 1st edition and by others); this method, therefore, is much saver to use than methods utilizing hazardous organic solvents, like phenol. The salt precipitation method produces exceptionally pure DNA as assessed in a spectrophotometer at A260/280 nm (readings of 1.7-2.0). Purified DNA can be stored for long periods of time (archival quality). In addition, large genomic DNA fragments (>100 kb) are purified, since no spin columns or harsh reagents are used. Therefore, the purified DNA can be used in a variety of downstream applications, like PCR, restriction digestion, Southern blot, sequencing and single nucleotide polymorphism (SNP) detection.
I have routinely used the Puregene Kit for isolation of genomic DNA from mouse tails in order to screen transgenic and knockout mouse colonies with PCR and Southern blots. For mouse tail screening, Qiagen (who recently purchased Gentra) now provides kits for isolation of 100 mg and 4 g of mouse tails. The kits include a cell lysis solution, a protein precipitation solution, a DNA hydration solution (10 mM Tris, 1 mM EDTA pH 7-8) and proteinase K (25 mg). If the optional RNase digestion step will be included in the protocol, the RNase A solution has to be purchased in addition. The kits, which are stable for 3 years, can be stored at room temperature, but the enzymes should be kept at 4¡ãC.
For the procedure, I used either the tips of mouse tails (0.6-0.8 cm long) or the ear punches obtained when I tagged the mice. Since downscaling the volumes of the kit is possible, I was able to use the same procedure with 1/3 to 1/2 of the volume of each solution (depending on the size of the ear pieces). With this method, I could isolate enough DNA even from one ear punch to run a mini-gel and 2-3 PCR reactions. During the isolation of DNA from ear punches, I could never see a DNA or protein pellet; therefore supernatants should be carefully removed through pipetting throughout the procedure. The PCR amplification result I obtained with earpiece-DNA was, in general, comparable with DNA from mouse tail; only sometimes, when more bands than the expected appeared, it was necessary to repeat the reactions with mouse tail DNA. The appearance of non-specific bands in PCR could be due to the use of too little DNA in the PCR reaction or contamination of RNA or protein in the DNA preparation. RNA or protein contamination sometimes also occurred during tail DNA isolation; therefore I routinely included the RNase digestion step in my protocol. Another possibility is to repurify the protein-contaminated DNA with a special protocol I obtained from Gentra after troubleshooting with technical service. Therefore, it is good to analyze the purity of the isolated DNA by spectrophotometer (A260/280 nm ¡Ü1.6 indicates contamination with protein, ¡Ý 2 RNA contamination). Since the amount of DNA isolated from earpieces was too low to measure in our spectrophotometer, I could only determine the purity in DNA isolated from tail.
I really liked to use this kit, especially when I had to process several samples (20-30) at the same time. To invert all of the samples simultaneously for mixing, I hold two flat multi-tube racks together with the tubes in one rack and the second rack on top of the tube lids. Using this mixing procedure, the DNA extraction can be limited to about 60-90 min after the initial overnight incubation.
I recommend this kit for the isolation of pure genomic DNA from not only different amounts of tissue but also from multiple samples at the same time. The protocol is easy to follow even for someone with little experience in molecular biology. For more information, there is a FAQ site on Gentra¡¯s website. In addition, Gentra¡¯s technical service was very helpful for troubleshooting the protein contamination problems; I expect this also from Qiagen¡¯s service.